EP4512819A2 - Zusammensetzungen und verfahren zur förderung der hypoxie oder der hypoxiereaktion zur behandlung und prävention von mitochondriendysfunktion und oxidativen stresserkrankungen - Google Patents

Zusammensetzungen und verfahren zur förderung der hypoxie oder der hypoxiereaktion zur behandlung und prävention von mitochondriendysfunktion und oxidativen stresserkrankungen Download PDF

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Publication number
EP4512819A2
EP4512819A2 EP24220669.6A EP24220669A EP4512819A2 EP 4512819 A2 EP4512819 A2 EP 4512819A2 EP 24220669 A EP24220669 A EP 24220669A EP 4512819 A2 EP4512819 A2 EP 4512819A2
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EP
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Prior art keywords
therapeutic gas
subject
hypoxia
disease
mitochondrial
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EP24220669.6A
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English (en)
French (fr)
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EP4512819A3 (de
Inventor
Vamsi K. Mootha
Isha JAIN
Warren M. Zapol
Luca ZAZZERON
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General Hospital Corp
Massachusetts Institute of Technology
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General Hospital Corp
Massachusetts Institute of Technology
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Publication of EP4512819A2 publication Critical patent/EP4512819A2/de
Publication of EP4512819A3 publication Critical patent/EP4512819A3/de
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Definitions

  • the present invention relates generally to compositions and methods that promote hypoxia or the hypoxia response for treating or preventing mitochondrial dysfunction and oxidative stress disorders.
  • mitochondrial disease pathology is thought to arise from an energy supply-demand imbalance.
  • redox state nucleotide biosynthesis
  • ROS homeostasis regulation of apoptosis
  • calcium signaling and fatty acid oxidation may be impaired in disease states.
  • mitochondrial disorders can be highly tissue-specific, and episodic ( Haas et al., Pediatrics. 120, 1326-33 (2007 )). Individuals with identical genetic lesions can follow completely distinct clinical trajectories. Such observations suggest that existing cellular pathways may buffer against lesions in unaffected tissues.
  • CRISPR genome-wide clustered regularly interspaced short palindrome repeats
  • FG-4592 treatment rewired energy metabolism, including an increase in the glycolytic capacity of cells, as well as a suppression of basal respiration.
  • FG-4592 treatment in vivo alleviated the sensitivity of zebrafish embryos to mitochondrial dysfunction.
  • the mouse model of mitochondrial disease evaluated herein is characterized by excess oxidative stress, indicating that reducing oxygen availability (and thus the availability of oxygen needed to produce reactive oxygen species) is an effective means to treat disorders characterized by excess oxidative stress.
  • the disclosure provides a method of treating or preventing mitochondrial dysfunction, an oxidative stress disorder, or an inflammatory disorder in a subject in need thereof comprising increasing the activity of a hypoxia response in the subject.
  • Increasing the activity of a hypoxia response can be achieved by, for example, exposing the subject to hypoxia.
  • the hypoxia response may include, but is not limited to, one or more of the following: a physiological response or a trigger of a hypoxia response.
  • the disclosure provides a method of treating or preventing mitochondrial dysfunction, an oxidative stress disorder, or an inflammatory disorder in a subject in need thereof, the method comprising administering to the subject by inhalation a therapeutically effective amount of a therapeutic gas at normobaria comprising between 5 to 20% O 2 .
  • the therapeutic gas comprises between 10 to 15% O 2 , between 10 to 12% O 2 , or about 11% O 2 .
  • the disclosure provides a method of treating or preventing mitochondrial dysfunction, an oxidative stress disorder, or an inflammatory disorder in a subject in need thereof, the method comprising causing the subject to breathe a therapeutically effective amount of air in a hypobaric chamber.
  • the hypobaric chamber has an atmospheric pressure equal to the atmospheric pressure at an elevation between 1,500 to 10,000 meters above sea level (e.g., an atmospheric pressure equal to the atmospheric pressure at an elevation between 1,500 to 8,000 meters or between 2,000 to 4,500 meters above sea level).
  • the disclosure provides a method of increasing the activity of a hypoxia response in a subject in need thereof comprising increasing the stability or the activation of HIF proteins in the subject.
  • the disclosure provides a treating or preventing mitochondrial dysfunction, an oxidative stress disorder, or an inflammatory disorder in a subject in need thereof comprising increasing cellular glycolysis in the subject.
  • the disclosure provides a method of treating or preventing mitochondrial dysfunction, an oxidative stress disorder, or an inflammatory disorder in a subject in need thereof comprising suppressing cellular basal respiration in the subject.
  • nitric oxide is administered in combination with a method described herein.
  • the therapeutic gas comprises nitric oxide (e.g., wherein the concentration of nitric oxide in the therapeutic gas is at least 5 ppm, at least 10 ppm, at least 20 ppm, or is in the range of 0.5 ppm to 80 ppm).
  • xenon is administered in combination with a method described herein.
  • the therapeutic gas comprises xenon (e.g., wherein the therapeutic gas comprises between 20-70% xenon).
  • an agent that reduces pulmonary hypertension or raises the cGMP level in other cells e.g., a phosphodiesterase inhibitor or a soluble guanylate cyclase sensitizer
  • a phosphodiesterase inhibitor or a soluble guanylate cyclase sensitizer is administered either systemically or by inhalation to the lung in combination with a method described herein.
  • phosphodiesterase inhibitors examples include: Zaprinast ® (M&B 22948; 2-o-propoxyphenyl-8-azapurine-6-one; Rhone-Poulenc Rorer, Dagenham Essex, UK);
  • WIN 58237 (1-cyclopentyl-3-methyl-6-(4-pyridyl)pyrazolo[3,4-d]pyrimidin-4-(5H)-one; Silver et al. (1994) J. Pharmacol. Exp. Ther. 271:1143 ); SCH 48936 ((+)-6a,7,8,9,9a,10,11,11a-octahydro-2,5-dimethyl-3H-pentalen(6a,1,4,5)imidazo[2,1-b]purin-4(5H)-one; Chatterjee et al.
  • Examples of compounds that sensitize soluble guanylate cyclase include: 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole ("YC-1"; Russwurm (2002) J. Biol. Chem. 277:24883 ; Schmidt et al. (2001) Mol. Pharmacol. 59:220 ; and Friebe et al. (1998) Mol. Pharmacol. 54:962 ); compounds loosely based on YC-1 such as the pyrazolopyridine BAY 41-2272 ( Stasch et al.
  • the therapeutic gas is administered to the subject continuously (e.g., for at least three minutes, at least 15 minutes, at least one hour, at least eight hours, or at least 24 hours). In some embodiments, the therapeutic gas is administered to the subject intermittently.
  • the therapeutic gas is humidified and administered to the subject by nasal prongs, a face mask, an enclosed tent or chamber, an intra-tracheal catheter, an endotracheal tube, or a tracheostomy tube.
  • the therapeutic gas can be administered to the subject by a tent that is positioned over a bed or a crib on which the subject is placed.
  • arterial oxygen saturation is measured in the subject one or more times after administration of the therapeutic gas to the subject (e.g., continuously during administration of the therapeutic gas to the subject); and/or arterial partial oxygen pressure (PaO 2 ) is measured in the subject one or more times after administration of the therapeutic gas to the subject (e.g., continuously during administration of the therapeutic gas to the subject).
  • the measured SpO 2 value is used to feedback and automatically determine the concentration of inspired oxygen so as to maintain SpO 2 in the subject in the range of 50-90%; and/or the measured PaO 2 value is used to feedback and automatically determine the concentration of inspired oxygen so as to maintain PaO 2 in the subject in the range of 25 mm Hg to 70 mm Hg.
  • the subject optionally has a mitochondrial disorder.
  • the mitochondrial disorder is in some examples a monogenic mitochondrial disorder.
  • the mitochondrial disorder is characterized by a mutation in a gene selected from the group consisting of AARS2, AASS, ABAT, ABCB6, ABCB7, ABCD1, ACACA, ACAD8, ACAD9, ACADM, ACADS, ACADSB, ACADVL, ACAT1, ACO2, ACSF3, ACSL4, ADCK3, ADCK4, AFG3L2, AGK, AGXT, AIFM1, AK2, ALAS2, ALDH18A1, ALDH2, ALDH3A2, ALDH4A1, ALDH5A1, ALDH6A1, ALDH7A1, AMACR, AMT, APOPT1, ATIC, ATP5A1, ATP5E, ATP6, ATP8, ATPAF2, ATXN2, AUH, BAX, BCKDHA, BCKDHB, BCKDK, BCS1L, BOLA3, C10orf2, C12orf65, CA5A, CARS2, CASP8, CAT, CEP89, CHCHD10, CISD2, CLPB,
  • the mitochondrial disorder is characterized by a biochemical deficiency of respiratory chain Complex I, II, III, IV, V, or a combination thereof.
  • the mitochondrial disorder presents with one or more of gray matter disease, white matter disease, seizures, migraines, ataxia, stroke, stroke-like episodes, deafness, optic neuropathy, peripheral neuropathy, retinopathy, external opthalmoplegia, liver failure, kidney failure, pancreatic exocrine dysfunction, intestinal pseudoobstruction, anemia, skeletal muscle myopathy, cardiomyopathy, cardiac conduction defects, short stature, hypogonadism, immune dysfunction, or metabolic acidosis.
  • the mitochondrial disorder is a mitochondrial respiratory chain disorder.
  • the subject is less than five years of age (e.g., less than one year of age).
  • the subject optionally has an age-associated disorder (e.g., type 2 diabetes, insulin resistance, neurodegeneration, peripheral neuropathy, sarcopenia, muscle atrophy, deafness, atherosclerosis, cardiovascular disease, heart failure, chronic kidney disease, cancer, arthritis, cataracts, or osteoporosis).
  • an age-associated disorder e.g., type 2 diabetes, insulin resistance, neurodegeneration, peripheral neuropathy, sarcopenia, muscle atrophy, deafness, atherosclerosis, cardiovascular disease, heart failure, chronic kidney disease, cancer, arthritis, cataracts, or osteoporosis.
  • the subject can be treated to prevent (completely or partially) the occurrence of mitochondrial dysfunction associated with aging.
  • the subject can be, for example, at least 20 years of age, at least 30 years of age, at least 40 years of age, or older.
  • the subject can benefit from treatment even without having any evident disease.
  • a subject can be administered by inhalation a therapeutically effective amount of a therapeutic gas comprising (i) between 5 to 20% O 2 , and (ii) nitric oxide (e.g., an amount of nitric oxide disclosed herein).
  • a subject can breathe a therapeutically effective amount of air in a hypobaric chamber in combination with inhalation of nitric oxide (e.g., an amount of nitric oxide disclosed herein).
  • the subject optionally exhibits mitochondrial dysfunction associated with aging (e.g., the subject is at least 65 years of age or is at least 75 years of age).
  • the mitochondrial dysfunction occurs in response to an environmental insult (e.g., a drug, an antibiotic, an antiviral drug, or a pesticide that is toxic to mitochondria.
  • an environmental insult e.g., a drug, an antibiotic, an antiviral drug, or a pesticide that is toxic to mitochondria.
  • the subject can be been identified as having a genetic mutation associated with onset of a mitochondrial disorder and treatment is initiated before the onset of symptoms of the disorder.
  • the subject can be identified as having a mutation in a gene selected from the group consisting of AARS2, AASS, ABAT, ABCB6, ABCB7, ABCD1, ACACA, ACAD8, ACAD9, ACADM, ACADS, ACADSB, ACADVL, ACAT1, ACO2, ACSF3, ACSL4, ADCK3, ADCK4, AFG3L2, AGK, AGXT, AIFM1, AK2, ALAS2, ALDH18A1, ALDH2, ALDH3A2, ALDH4A1, ALDH5A1, ALDH6A1, ALDH7A1, AMACR, AMT, APOPT1, ATIC, ATP5A1, ATP5E, ATP6, ATP8, ATPAF2, ATXN2, AUH, BAX, BCKDHA, BCKDHB, BCKDK, BCS1L,
  • oxidative stress disorders examples include Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, Asperger syndrome, attention deficit hyperactivity disorder, diabetes, cardiovascular disease, cancer, Lafora disease, atherosclerosis, heart failure, myocardial infarction, fragile X syndrome, sickle cell disease, lichen planus, vitiligo, and autism.
  • inflammatory disorders examples include rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), psoriasis, inflammatory myositis, Langerhans-cell histiocytosis, adult respiratory distress syndrome, Wegener's granulomatosis, vasculitis, cachexia, stomatitis, idiopathic pulmonary fibrosis, dermatomyositis, polymyositis, non-infectious scleritis, chronic sarcoidosis with pulmonary involvement, myelodysplastic syndrome, moderate to severe chronic obstructive pulmonary disease without significant right to left shunting of blood, and giant cell arteritis.
  • inflammatory bowel disease e.g., ulcerative colitis and Crohn's disease
  • psoriasis inflammatory myositis
  • the disclosure provides a system comprising (i) an enclosed tent or chamber or a breathing apparatus, (ii) a hypoxia induction system that delivers oxygen-depleted air to the enclosed tent or chamber or the breathing apparatus, wherein the oxygen-depleted air comprises between 5 to 20% O 2 , and (iii) a device (e.g., pulse oximeter) that measures arterial oxygen saturation in a subject breathing air within the enclosed tent or chamber or from the breathing apparatus, wherein the system adjusts the oxygen content of the oxygen-depleted air delivered to the enclosed tent or chamber or the breathing apparatus based upon the oxygen saturation measured by the device such that oxygen saturation in the subject is maintained within the range of 50% to 90% (e.g., within the range of 80% to 90% or at about 85%, or within the range of 55% to 65% or at about 80%).
  • a hypoxia induction system that delivers oxygen-depleted air to the enclosed tent or chamber or the breathing apparatus, wherein the oxygen-depleted air comprises between 5 to 20% O 2
  • a device e
  • the hypoxia induction system comprises a first container comprising a first gas comprising nitrogen and a second container comprising a second gas comprising oxygen, and wherein the oxygen-depleted air delivered to the enclosed tent or chamber or the breathing apparatus is prepared by mixing the first gas and the second gas.
  • the hypoxia induction system intakes ambient air, reduces the oxygen content of the intake air, to produce the oxygen-depleted air that is delivered to the enclosed tent or chamber or the breathing apparatus.
  • the hypoxia induction system intakes ambient air, adds nitrogen to the intake air, to produce the oxygen-depleted air that is delivered to the enclosed tent or chamber or the breathing apparatus.
  • the subject can be a human subject.
  • the disclosure provides a method of screening for a compound that increases the activity of a hypoxia response comprising
  • the disclosure provides a method of screening for targets for the modulation of mitochondrial respiratory chain function comprising
  • the disclosure provides a method of treating or preventing mitochondrial dysfunction, an oxidative stress disorder, or an inflammatory disorder in a subject in need thereof comprising increasing the activity of the hypoxia response pathway in the subject.
  • the method comprises suppression of mitochondrial disease.
  • the suppression of mitochondrial disease occurs via mediation of the hypoxia response.
  • the disclosure also provides methods of screening for compounds that treat or prevent mitochondrial dysfunction, an oxidative stress disorder, or an inflammatory disorder.
  • methods of screening for a compound that increases the activity of a hypoxia response is provided.
  • methods of screening for targets for the modulation of response to mitochondrial respiratory chain dysfunction are provided.
  • hypoxia refers to a deficiency of oxygen.
  • a low oxygen condition is also referred to as a “hypoxic condition.” See FIG. 1 for a schematic representation of hypoxia. HIF1 ⁇ is stabilized during hypoxia.
  • hypoxia inducible transcription factor is an oxygen-sensitive transcription factor that responds to low oxygen.
  • hypoxia inducible transcription factors include alpha subunits of hypoxia inducible transcription factors (e.g., HIP1 ⁇ , HIF2 ⁇ and HIF3 ⁇ ), and beta subunits (HIF1 ⁇ , HIF2 ⁇ , and HIF3 ⁇ ).
  • HIFs are also referred to herein as HIF proteins.
  • HIF is a heterodimer comprising an alpha and a beta subunit, which induces transcription of HIF-responsive genes during hypoxia or under hypoxic conditions.
  • HIF-responsive genes include but are not limited to genes involved in glucose metabolism, for example, transport (e.g., glucose transporter 1 (GLUT1) and glucose transporter 3 (GLUT3)), tricarboxylic acid (TCA) cycle (also known as the Krebs cycle or the citric acid cycle, e.g., PDK1), glycolysis (e.g., hexokinase 1 (HK1); hexokinase 2 (HK2); glyceraldehyde 3-phosphate dehydrogenase (GAPDH); 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKBF3); 6-phosphofructo-2-kinase, liver type (PFKL); phosphoglycerate kinase 1 (PGK1); and pyruvate kinase, muscle (PKM)); redox modulation (e.g., lactate dehydrogenase A (LDHA) and monocarbox
  • hypoxia response is a response by a cell and/or an organism to hypoxia. Hypoxia is one non-limiting way to induce a hypoxia response.
  • a hypoxia response includes, but is not limited to, a physiological response (e.g., a systemic or pulmonary hemodynamic response, a change in the regulation of cellular metabolism, and up-regulation of genes (e.g., HIF responsive genes)) and a pathological response (e.g., pulmonary hypertension, cerebral ischemia, myocardial ischemia, and tumor angiogenesis).
  • a physiological response e.g., a systemic or pulmonary hemodynamic response, a change in the regulation of cellular metabolism, and up-regulation of genes (e.g., HIF responsive genes)
  • pathological response e.g., pulmonary hypertension, cerebral ischemia, myocardial ischemia, and tumor angiogenesis.
  • Non-limiting examples of systemic responses include pulmonary vasoconstriction, systemic vasodilation, increased cytosolic calcium concentration, and neurotransmitter release, for example, catecholamines, acetylcholine, and serotonin.
  • Non-limiting examples of a response affecting the regulation of cellular metabolism include uncontrolled cell swelling, cell necrosis, impaired mitochondrial respiratory chain function, increased cellular glycolysis, decreased cellular energy consumption, and decreased cellular oxygen consumption.
  • hypoxia response examples include increased ventilation, increased cardiac output, a switch from aerobic to anaerobic metabolism, promotion of improved vascularization, an increase of erythropoietin with augmented erythropoiesis, enhancement of the oxygen carrying capacity of the blood, reduced oxygen toxicity, increased or reduced reactive oxygen species, and increased or reduced oxidative stress.
  • a hypoxia response may involve oxygen-responsive pathways to sense and to respond to changes in oxygen availability.
  • HIFs may respond to a low oxygen environment and activate one or more HIF-responsive genes.
  • Normoxia or a "normoxic condition” refers to a normal level of oxygen condition. See FIG. 2 for a schematic representation of normoxia. HIF1 ⁇ is degraded under normoxic conditions.
  • PHD prolyl-hydroxylase
  • PHD enzymes hydroxylate alpha subunits of HIF at conserved proline residues. Hydroxylation and degradation occurs under normoxic conditions. PHD enzyme activity is inhibited under hypoxic conditions.
  • PHD inhibitors include 2-oxoglutarate analogs (also known as ⁇ -ketoglutarate, e.g., roxadustat, 2,4-diethylpyridine dicarboxylate, dimethyloxallyl glycine, IOX2, and N-oxalylglycine), ⁇ -oxocarboxylic acids (e.g., 1,4-dihydrophenonthrolin-4-one-3-carboxylic acid), and BAY-85-3934 (also known as 2-(6-morpholinopyrimidin-4-yl)-4-(1H-1,2,3-triazol-1-yl)-1,2-dihydro-3H-pyrazol-3-one).
  • Roxadustat is also known as FG-4592 and N-[(4-hydroxy-1-methyl-7-phenoxy-3-isoquinolinyl)carbonyl]glycine.
  • IOX2 is also known as (1-benzyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carbonyl)glycine.
  • Additional examples of 2-oxoglutarate analogs as PHD inhibitors include 4-hydroxyisoquinoline-2-carbonylglycine derivatives, 4-hydroxy-2-quinoline, pyrrolopyridines, thiazolopyridines, isothiazolopyridines, 4-hydroxycoumarins, and 4-hydroxythiocoumarins ( 11 ).
  • FG-2216 ((1-chloro-4-hydroxyisoquinoline-3-carbonyl)glycine) and FG-4497 ((1-hydroxy-6-(phenylthio)isoquinoline-3-carbonyl)glycine).
  • Any known prolyl-hydroxylase inhibitor may be used in methods of the invention.
  • PHD inhibitors are disclosed in M. Rabinowitz, Inhibition of hypoxia-inducible factor prolyl hydroxylase domain oxygen sensors: tricking the body into mounting orchestrated survival and repair responses. J. Med. Chem. 56, 9369-9402 (2013 ), incorporated herein by reference in its entirety.
  • VHL Von Hippel Lindau
  • endogenous cellular oxygen sensing involves a mechanism or process used by the body or cells to determine or to measure the levels of oxygen available to the cells. For example, oxygen sensing may occur using a hypoxia response.
  • mitochondrial respiratory chain disorder is a heterogeneous group of genetic disorders that share involvement of the cellular bioenergetics machinery due to molecular defects affecting the mitochondrial oxidative phosphorylation system. Over 150 different genetic causes of mitochondrial respiratory chain disorder impact one or more of the five respiratory chain complexes.
  • the respiratory chain complexes include Complex I (NADH-coenzyme Q reductase or NADH dehydrogenase), Complex II (succinate-coenzyme Q reductase or succinate dehydrogenase), Complex III (cytochrome bc 1 complex or coenzyme Q-cytochrome C oxidoreductase), Complex IV (cytochrome C oxidase), and Complex V (ATP synthase, adenosine triphosphate synthase).
  • a mitochondrial lesion is damage to a gene encoded by mitochondrial DNA or nuclear-encoded mitochondrial protein. The mitochondrial lesion may be introduced by oxidative stress. Mitochondrial diseases include a mitochondrial respiratory chain disorder.
  • a “Complex I inhibitor” inhibits the functioning of the NADH-coenzyme Q reductase in the mitochondrial electron transport chain and prevents electron transfer from NADH to coenzyme Q10.
  • Non-limiting examples of a Complex I inhibitor include acetogenins (e.g., annonacin, bullatacin or rolliniastatin-2, and uvaricin), reduced nicotinamide adenine dinucleotide (NADH) analogs (e.g., adenosine diphosphate ribose), ubiquinone analogs (e.g., piericidin (also referred to as piericidin A) and rotenone), and metformin.
  • NADH reduced nicotinamide adenine dinucleotide
  • ubiquinone analogs e.g., piericidin (also referred to as piericidin A) and rotenone
  • metformin metformin.
  • a “Complex III inhibitor” inhibits the functioning of the coenzyme Q-cytochrome C oxidoreductase in the mitochondrial electron transport chain and prevents the biochemical generation of ATP.
  • a Complex III inhibitor include Q i site inhibitors (e.g., antimycin) and Q o site inhibitors (quinone outside inhibitors, e.g., myxothiazol, stigmatellin, and strobilurin derivatives).
  • a “Complex V inhibitor” inhibits the functioning of ATP synthase in the mitochondrial electron transport chain and prevents the biochemical generation of ATP.
  • a Complex V inhibitor include ⁇ -helical basic peptide inhibitors (e.g., melittin), catechins (e.g., epicatechin, epicatechin gallate, and epigallocatechin gallate), catecholestrogens (e.g., 4-hydroxyestradiol and 2-hydroxyestradiol), flavones (e.g., quercetin, morin, kaempferol, and genistein), oligomycins (e.g., oligomycin A and oligomycin), polyketide inhibitors (e.g., peliomycin, venturicidin A, B, and X, and ossamycin), stilbenes (e.g., resveratrol, piceatannol, and diethylstilbestrol), tentoxin and derivatives (e.g.,
  • a "compromised function of the mitochondrial respiratory chain” refers to one or more cells with abnormal functioning of the cellular bioenergetics machinery affecting the mitochondrial oxidative phosphorylation system. For example, this may be a result of genetic defects in one or more respiratory chain complexes, a mitochondrial lesion, an inhibitor of one or more respiratory chain complexes or any physiological situation which impairs mitochondrial respiratory chain function.
  • basal respiration refers to routine respiration in an intact and healthy cell.
  • the proton current generated by basal respiration supplies ATP synthesis and the proton leak.
  • cellular stress may be caused by environmental stressors including temperature extremes, toxin exposure, mechanical damage, and hypoxic conditions. For example, viral prodromes, dehydration, and low oxygen cause cellular stress. When a cell is exposed to unfavorable environmental conditions of cellular stress, the cell can mount a response to protect the cell against the environmental stressors.
  • a "high energy demand” requires the cell to produce a large amount of energy to perform required functions.
  • mitochondria are organelles that carry out the process of aerobic respiration to breakdown molecules like glucose in the presence of oxygen.
  • the cellular bioenergetics machinery uses respiratory chain complexes to produce energy for a cell.
  • high energy demand occurs in cells when an organism comprising those cells exerts itself beyond a sedentary condition.
  • high energy demand occurs when an organism comprising the cells is injured in a location near the cells.
  • high energy demand occurs when an organism comprising the cells is suffering from an infectious disease that affects the cells.
  • an “age-associated disorder” is a disease or a disorder seen with increasing frequency as individuals age. These disorders are associated with gradual deterioration of function (e.g., quantitative decline in the activity of the mitochondrial respiratory chain).
  • Non-limiting examples of an age-associated disorder include type 2 diabetes, neurodegeneration (e.g., Alzheimer's disease), sarcopenia (muscle loss), atherosclerosis, cardiovascular disease, cancer, arthritis, cataracts, and osteoporosis.
  • CRISPR is a clustered regularly interspaced short palindrome repeat guided by RNA to introduce a targeted loss-of function mutation at one or more specific sites in the genome.
  • the system includes a sgRNA and an endonuclease such as the CRISPR associated protein 9 (Cas9) nuclease.
  • Cas9 CRISPR associated protein 9
  • Various compositions and methods of use related to the delivery, engineering, optimization and therapeutic applications of systems, methods, and compositions used for the control of gene expression involving sequence targeting, such as genome perturbation or gene-editing, may be utilized in the invention.
  • the genome perturbation or gene-editing relates to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the CRISPR-Cas system does not require the generation of customized proteins to target specific sequences but rather a single Cas enzyme can be programmed by a short RNA molecule to recognize a specific DNA target.
  • Examples of useful CRISPR-Cas systems and components include, but are not limited to, the components, or any corresponding orthologs thereof, and delivery of such components, including methods, materials, delivery vehicles, vectors, particles, AAV, and making and using thereof, as described in, e.g., US Patents Nos.
  • RNAi Gene Enrichment Ranking uses an algorithm to rank screening hits by the consistent enrichment among multiple sgRNAs targeting the same gene.
  • the highest ranking (e.g., lowest number, rank 1) gene represents a gene target. Additional details about the algorithm can be found in B. Luo et al., Highly parallel identification of essential genes in cancer cells. Proc. Natl. Acad. Sci. U.S.A. 105, 20380-20385 (2008 ), incorporated herein by reference in its entirety.
  • a “sgRNA” is an RNA that guides the insertion or deletion of nucleotides into target locations in concert with the Cas9 nuclease.
  • the current disclosure provides a method of treating or preventing mitochondrial dysfunction, an oxidative stress disorder, or an inflammatory disorder in a subject in need thereof comprising increasing the activity of a hypoxia response in the subject.
  • Increasing the activity of a hypoxia response can be achieved by, for example, exposing the subject to hypoxia.
  • the hypoxia response may include, but is not limited to, one or more of the following: a physiological response (e.g., a systemic response, a change in the regulation of cellular metabolism, and up-regulation of genes (e.g., HIF responsive genes)) or a trigger of a hypoxia response (e.g., cerebral ischemia, myocardial ischemia, and tumor angiogenesis).
  • hypoxia response is a systemic or pulmonary response selected from the group consisting of pulmonary vasoconstriction, systemic vasodilation, increased cytosolic calcium concentration, neurotransmitter release.
  • hypoxia response is a response affecting the regulation of cellular metabolism selected from the group consisting of uncontrolled cell swelling, cell necrosis, impaired mitochondrial respiratory chain function, increased cellular glycolysis, and decreased cellular energy consumption.
  • a hypoxia response is selected from the group consisting of increased ventilation, increased cardiac output, a switch from aerobic to anaerobic metabolism, promotion of improved vascularization, and augmented erythropoietin levels with enhancement of erythropoiesis, enhancement of the oxygen carrying capacity of the blood, reduced oxygen toxicity, increased or reduced reactive oxygen species, and increased or reduced oxidative stress.
  • increasing the activity of a hypoxia response includes increasing the activation of a hypoxia inducible transcription factor (HIF).
  • HIF is selected from the group consisting of alpha or beta subunits of hypoxia inducible transcription factors.
  • the HIF is selected from the group consisting of HIF1 ⁇ , HIF3 ⁇ , HIF1 ⁇ , HIF2 ⁇ , HIF3 ⁇ , and HIF2 ⁇ .
  • increasing the activity of a hypoxia response includes inducing transcription of HIF-responsive genes.
  • the transcribed gene is involved in one of the following glucose metabolism, glucose transport, glycolysis, redox modulation, feedback regulation, angiogenesis, promoting red blood cell maturation and oxygen transport, erythropoiesis, and iron metabolism.
  • the HIF-responsive gene is selected from the group consisting of glucose transporter 1 (GLUT1); glucose transporter 3 (GLUT3); pyruvate dehydrogenase kinase, isozyme 1 (PDK1); hexokinase 1 (HK1); hexokinase 2 (HK2); glyceraldehyde 3-phosphate dehydrogenase (GAPDH); 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKBF3); 6-phosphofructo-2-kinase, liver type (PFKL); phosphoglycerate kinase 1 (PGK1); pyruvate kinase, muscle (PKM); lactate dehydrogenase A (LDHA); monocarboxylate transporter 4 (MCT4); Egl-9 family hypoxia-inducible factor 1 (EGLN1); Egl-9 family hypoxia-inducible factor
  • increasing the activity of a hypoxia response is done during normoxic conditions. In some embodiments, increasing the activity of a hypoxia response is not through stabilization or activation of HIF1 ⁇ . In still another embodiment, increasing the activity of a hypoxia response comprises bypassing endogenous cellular oxygen sensing.
  • the subject comprises mitochondria comprising one or more mitochondrial lesions or other lesions which impact mitochondrial respiratory chain function.
  • the one or more mitochondrial lesions may be introduced by oxidative stress.
  • the one or more mitochondrial lesions occur in a respiratory chain complex.
  • the one or more mitochondrial lesions occur in a protein complex, wherein the protein complex is selected from the group consisting of: Complex I, Complex II, Complex III, Complex IV, Complex V, and ATP (adenosine triphosphate) Synthase.
  • the method comprises inhibition of one or more proteins involved in the hypoxia response. In certain embodiments, inhibition of one or more proteins involved in a hypoxia response increases the activity of the hypoxia response. In some embodiments, the method comprises inhibition of PHD or VHL protein.
  • the PHD inhibitor is selected from the group consisting of 2-oxoglutarate analogs (also known as ⁇ -ketoglutarate), ⁇ -oxocarboxylic acids, and BAY-85-3934. In some embodiments, the PHD inhibitor is FG-4592 or roxadustat.
  • One of skill in the art may use any PHD inhibitor or VHL inhibitor known in the art for use in methods disclosed herein.
  • the method comprises increasing the stability or the activation of hypoxia inducible transcription factor (HIF).
  • increasing the activity of the hypoxia response comprises increasing the stability or the activation of HIF proteins.
  • increasing the stability of HIF proteins is done during normoxic conditions.
  • the mitochondrial disorder is a genetic disorder that affects the mitochondrial oxidative phosphorylation system. In some embodiments, the mitochondrial disorder affects one or more of the five respiratory chain complexes. In certain embodiments, the respiratory chain complexes is selected from the group consisting of Complex I (NADH-coenzyme Q reductase or NADH dehydrogenase), Complex II (succinate-coenzyme Q reductase or succinate dehydrogenase), Complex III (cytochrome bc 1 complex or ubiquinone-cytochrome C oxidoreductase), Complex IV (cytochrome C oxidase), and Complex V (ATP synthase).
  • Complex I NADH-coenzyme Q reductase or NADH dehydrogenase
  • Complex II succinate-coenzyme Q reductase or succinate dehydrogenase
  • Complex III cytochrome bc 1 complex or ubiquinone-cytochrome C oxidoreduct
  • the subject has an age-associated disorder.
  • the age-associated disorder is selected from the group consisting of type 2 diabetes, neurodegeneration (e.g., Alzheimer's disease), sarcopenia (muscle loss), insulin resistance, peripheral neuropathy, muscle atrophy, deafness, atherosclerosis, cardiovascular disease, heart failure, chronic kidney disease, cancer, arthritis, cataracts, and osteoporosis.
  • the age-associated disorder is selected from the group consisting of type 2 diabetes, neurodegeneration and sarcopenia.
  • the current disclosure provides a method of increasing the activity of a hypoxia response in a subject in need thereof comprising increasing the stability or the activation of HIF proteins in the subject.
  • the current disclosure provides a method of treating or preventing mitochondrial dysfunction, an oxidative stress disorder, or an inflammatory disorder in a subject in need thereof comprising increasing cellular glycolysis in the subject.
  • increasing cellular glycolysis comprises activation of a gene involved in glycolysis.
  • the gene involved in glycolysis is selected from the group consisting of hexokinase 1 (HK1); hexokinase 2 (HK2); glyceraldehyde 3-phosphate dehydrogenase (GAPDH); 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (PFKBF3); 6-phosphofructo-2-kinase, liver type (PFKL); phosphoglycerate kinase 1 (PGK1); and pyruvate kinase, muscle (PKM).
  • increasing cellular glycolysis is done during normoxic conditions.
  • increasing cellular glycolysis comprises bypassing endogenous cellular oxygen sensing.
  • the current disclosure provides a method of treating or preventing mitochondrial dysfunction, an oxidative stress disorder, or an inflammatory disorder in a subject in need thereof comprising suppressing cellular basal respiration in the subject.
  • the suppressing cellular basal respiration is done during normoxic conditions.
  • suppressing cellular basal respiration comprises bypassing endogenous cellular oxygen sensing.
  • the treatment using one of the methods disclosed herein is during a period of cellular stress.
  • the period of cellular stress corresponds to hypoxic conditions.
  • the treatment using one of the methods disclosed herein is during a period of high energy demand.
  • the period of high energy demand uses respiratory chain complexes to produce energy for a cell.
  • the method of treatment or prevention of mitochondrial dysfunction, an oxidative stress disorder, or an inflammatory disorder in a subject in need thereof is applied over a period of time that can range, e.g., from 8hrs/day (sleep period) to about 1 day to about 50 years, and more usually 1 week to about 25 years (e.g., 3 months, 6 months, 1 year, 5 years, and 10 years).
  • the treatment using one of the methods disclosed herein occurs over time and is a chronic treatment.
  • the method of treatment reduces or treats one or more symptoms of the mitochondrial disease.
  • symptoms mays include loss of motor control (e.g., ataxia (abnormal muscle coordination), dystrophic posturing, involuntary movements, and myoclonus), muscle weakness and pain (e.g., dystonia, hypotonia, lethargy, and myopathy), gastro-intestinal disorders and swallowing difficulties, poor growth, cardiac disease, liver disease, diabetes, respiratory complications (e.g., respiratory failure), seizures, dementia, coma, visual problems (e.g., eye muscle paralysis, nystagmus, ophthalmoplegia, optic atrophy, and pigmentary retinopathy (retinal color changes with loss of vision)), hearing problems (e.g., hearing loss), sensory neuropathy (nerve damage involving the sense organs), lactic acidosis, developmental delays and susceptibility to infection.
  • motor control e.g., ataxia (abnormal muscle coordination), dystrophic posturing, involuntary movements
  • mitochondrial disease causes cell injury or cell death of cells in the brain, heart, liver, skeletal muscles, kidneys, endocrine system, and respiratory system.
  • a mitochondrial disorder involves a deficiency in one or more respiratory chain complexes including Complex I (NADH-coenzyme Q reductase or NADH dehydrogenase), Complex II (succinate-coenzyme Q reductase or succinate dehydrogenase), Complex III (cytochrome bc 1 complex or ubiquinone-cytochrome C oxidoreductase), Complex IV (cytochrome C oxidase), and Complex V (ATP synthase).
  • a mitochondrial disorder involves one or more of the following diseases myopathy (muscle disease), mitochondrial encephalomyopathy (brain and muscle disease), fatal infantile multisystem disorder.
  • a mitochondrial disorder is characterized by a mutation in a gene selected from the group consisting of AARS2, AASS, ABAT, ABCB6, ABCB7, ABCD1, ACACA, ACAD8, ACAD9, ACADM, ACADS, ACADSB, ACADVL, ACAT1, ACO2, ACSF3, ACSL4, ADCK3, ADCK4, AFG3L2, AGK, AGXT, AIFM1, AK2, ALAS2, ALDH18A1, ALDH2, ALDH3A2, ALDH4A1, ALDH5A1, ALDH6A1, ALDH7A1, AMACR, AMT, APOPT1, ATIC, ATP5A1, ATP5E, ATP6, ATP8, ATPAF2, ATXN2, AUH, BAX, BCKDHA, BCKDHB, BCKDK, BCS1L, BOLA3, C10orf2, C12orf65, CA5A, CARS2, CASP8, CAT, CEP89, CHCHD10, CISD2, CL
  • a mitochondrial disorder is characterized by a point mutation in the mitochondrial DNA (mtDNA), deletion within the mtDNA, duplication within the mtDNA, or depletion of the mtDNA.
  • mtDNA mitochondrial DNA
  • a mitochondrial disorder is Kearns-Sayre syndrome (KSS), Leber's hereditary optic neuropathy (LHON), myoclonic epilepsy ragged red fiber syndrome (MERRF), mitochondrial encephalopathy, lactic acidosis, and stroke (MELAS) syndrome, sensory ataxic neuropathy, dysarthria, and ophthalmoparesis (SANDO) syndrome, maternally inherited Leigh syndrome (MILS), myopathy and external ophthalmoplegia, neuropathy, gastrointestinal encephalopathy (MNGIE) syndrome, Leigh syndrome, maternally inherited diabetes and deafness (MIDD) syndrome, Alpers-Huttenlocher syndrome, Sengers syndrome, mitochondrial myopathy, lactic acidosis and sideroblastic anemia (MLASA), chronic progressive external ophthalmoplegia (CPEO), autosomal dominant progressive external ophthalmoplegia (AdPEO), neuropathy, ataxia, retinitis pigmentosa
  • KSS
  • a mitochondrial disorder presents with one or more of gray matter disease, white matter disease, seizures, migraines, ataxia, stroke, stroke-like episodes, deafness, optic neuropathy, peripheral neuropathy, retinopathy, external opthalmoplegia, liver failure, kidney failure, pancreatic exocrine dysfunction, intestinal pseudoobstruction, anemia, skeletal muscle myopathy, cardiomyopathy, cardiac conduction defects, short stature, hypogonadism, immune dysfunction, or metabolic acidosis.
  • the mitochondrial disorder is diagnosed by an algorithm selected from the group consisting of the Bernier criteria ( Bernier et al., “Diagnostic criteria for respiratory chain disorders in adults and children," Neurology, 59(9):1406-11, 2002 ), the Morava criteria ( Morava et al., “Mitochondrial disease criteria: diagnostic applications in children," Neurology, 67(10):1823-6, 2006 ), and Consensus from the Mitochondrial Medicine Society ( Parikh et al., "Diagnosis and management of mitochondrial disease: a consensus statement from the Mitochondrial Medicine Society," Genetics in Medicine, 17(9):689-701, 2015 ).
  • a subject exhibits mitochondrial dysfunction associated with aging.
  • the subject can be, e.g., at least 65 years of age, at least 70 years of age, at least 75 years of age, or at least 80 years of age.
  • the subject is treated to prevent (completely or partially) the occurrence of mitochondrial dysfunction associated with aging.
  • the subject can be, e.g., at least 15 years of age, at least 20 years of age, at least 25 years of age, at least 30 years of age, at least 35 years of age, or at least 40 years of age.
  • the subject in these embodiments need not exhibit mitochondrial dysfunction or other form of evident disease.
  • a subject exhibits mitochondrial dysfunction that occurs in response to an environmental insult such as ingestion of antibiotics (e.g., tetracycline, chloramphenicol, or aminoglycosides), antivirals (e.g., stavudine), pesticides (e.g., rotenone), or licit drugs (e.g., MPTP) that are toxic to mitochondria.
  • antibiotics e.g., tetracycline, chloramphenicol, or aminoglycosides
  • antivirals e.g., stavudine
  • pesticides e.g., rotenone
  • licit drugs e.g., MPTP
  • a subject has an oxidative stress disorder.
  • oxidative stress disorders include Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, multiple sclerosis, Asperger syndrome, attention deficit hyperactivity disorder, diabetes, cardiovascular disease, cancer, Lafora disease, atherosclerosis, heart failure, myocardial infarction, fragile X syndrome, sickle cell disease, lichen planus, vitiligo, and autism.
  • a subject has an inflammatory disorder.
  • inflammatory disorders include rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease), psoriasis, inflammatory myositis, Langerhans-cell histiocytosis, adult respiratory distress syndrome, Wegener's granulomatosis, vasculitis, cachexia, stomatitis, idiopathic pulmonary fibrosis, dermatomyositis, polymyositis, non-infectious scleritis, chronic sarcoidosis with pulmonary involvement, myelodysplastic syndrome, moderate to severe chronic obstructive pulmonary disease, and giant cell arteritis.
  • the current disclosure provides a method of screening for a compound that increases the activity of a hypoxia response comprising
  • cell growth is stunted or arrested when respiratory chain complexes are inhibited for most cell lines.
  • inhibition of one of the following respiratory chain complexes: Complex I, Complex III, or Complex V stunts cell growth in most cell lines.
  • inhibition of Complex I, Complex III, or Complex V stops cell growth in a cell line.
  • the methods of screening disclosed herein use one or more cells that are HT-29 cells, HEK 293 cells, or K562 cells.
  • a compromised function of the mitochondrial respiratory chain comprises the presence of genetic defects in one or more respiratory chain complexes, triggering a mitochondrial lesion, or administering an inhibitor of one or more respiratory chain complexes.
  • the compromised function of the mitochondrial respiratory chain comprises an inhibitor of one or more respiratory chain complexes.
  • the compromised function of the mitochondrial respiratory chain comprises administration of antimycin to the one or more cells in the presence of pyruvate.
  • the compromised function of the mitochondrial respiratory chain further comprises the reduction of or removal of pyruvate.
  • cell growth is reduced by administering a Complex I inhibitor, a Complex III inhibitor, or a Complex V inhibitor.
  • the compromised function of the mitochondrial respiratory chain comprises an inhibition selected from the group consisting of Complex III Inhibition, Complex I Inhibition, Complex V Inhibition, and ATP (adenosine triphosphate) Synthase Inhibition.
  • the compromised function of the mitochondrial respiratory chain comprises administering a Complex I inhibitor, a Complex III inhibitor, or a Complex V inhibitor.
  • the Complex I inhibitor is selected from the group consisting of acetogenins, reduced nicotinamide adenine dinucleotide (NADH) analogs, and metformin.
  • the Complex I inhibitor is selected from the group consisting of annonacin, bullatacin, rolliniastatin-2, uvaricin, adenosine diphosphate ribose, piericidin, rotenone, and metformin.
  • the Complex III inhibitor is selected from the group consisting of Q i site inhibitors and Q o site inhibitors.
  • the Complex III inhibitor is selected from the group consisting of Antimycin, myxothiazol, stigmatellin, and strobilurin derivatives.
  • the ATP synthase inhibitor is selected from the group consisting of oligomycins, ⁇ -helical basic peptide inhibitors, catechins, catecholestrogens, flavones, polyketide inhibitors, stilbenes, tentoxin and derivatives, and nucleotide analogs.
  • the ATP synthase inhibitor is selected from the group consisting of oligomycin, melittin, epicatechin, epicatechin gallate, epigallocatechin gallate, 4-hydroxyestradiol, 2-hydroxyestradiol, quercetin, morin, kaempferol, genistein, peliomycin, venturicidin A, venturicidin B, venturicidin X, ossamycin, resveratrol, piceatannol, diethylstilbestrol, tentoxin, GTP, FTP, and TNP-ATP.
  • the current disclosure provides a method of screening for targets for the modulation of mitochondrial respiratory chain function by genetic engineering techniques.
  • the genetic engineering technology comprises a recombinant CRISPR-Cas expression system.
  • the Cas protein is complexed with a sgRNA as provided herein and in the art.
  • the method of screening for targets for modulation of mitochondrial respiratory chain function comprising
  • compromising the function of the mitochondrial respiratory chain comprises introducing genetic defects in one or more respiratory chain complexes, triggering a mitochondrial lesion, or administering an inhibitor of one or more respiratory chain complexes.
  • the compromise of function of the mitochondrial respiratory chain comprises administration of an inhibitor of one or more respiratory chain complexes.
  • the compromise of function of the mitochondrial respiratory chain comprises administration of antimycin to the one or more cells in the presence of pyruvate.
  • the compromise of function of the mitochondrial respiratory chain further comprises the reduction of or removal of pyruvate.
  • composition comprising a nucleic acid molecule comprising a sequence selected from the group consisting of:
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount required by the methods of treatment.
  • the physician or veterinarian could start doses of the inhibitors or sequences of the invention employed in the methods at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • the specific dose level and frequency of dosage for any particular patient or subject may be varied and will depend upon a variety of factors including the efficacy of the specific method employed, the metabolic stability and length of action of that method, the age, body weight, general health, gender, diet, and the severity of the particular mitochondrial disorder being treated.
  • specific dose level and frequency of dosage for any particular patient also may depend on factors including, but not limited to, other medications, allergies, and prior experiences with treatments. For example, some patients may be treated using methods of this disclosure over a period of years if the mitochondrial disorder is a chronic condition.
  • kits are provided.
  • the kit includes a container for the screening assay.
  • An instruction for the use of the assay and the information about the screening method are to be included in the kit.
  • the kit may also contain a Complex I inhibitor, a Complex III inhibitor, or a Complex V inhibitor to mimic a mitochondrial respiratory chain disorder.
  • the kit may also contain sgRNAs having a nucleic acid sequence comprising one or more sequences targeting at least one gene in the human genome.
  • Mitochondrial dysfunction, an oxidative stress disorder, or an inflammatory disorder can be treated or prevented by administering to a subject by inhalation a therapeutically effective amount of a therapeutic gas comprising between 5 to 20% O 2 .
  • a hypoxia induction system can be used to prepare the therapeutic gas that is administered to the subject.
  • a hypoxia induction system contains a first container containing a first gas comprising nitrogen and a second container containing a second gas comprising oxygen, and the oxygen-depleted air delivered to the subject is prepared by mixing the first gas and the second gas.
  • a hypoxia induction system intakes ambient air, reduces the oxygen content of the intake air, to produce the oxygen-depleted air that is delivered to the subject.
  • Colorado Altitude Training (CAT) provides a portable enclosure system that produces hypoxic air by this mechanism.
  • CAT uses a high-flow hypoxic air delivery unit and a semi-sealed enclosure. The air unit draws in ambient room air and separates the oxygen molecules from the nitrogen molecules, creating the hypoxic or oxygen reduced air.
  • Simulated altitudes of up to 14,500 feet can be achieved, and can be controlled to within 100 feet accuracy using CAT's digital control system.
  • Hypoxico Altitude Training Systems devices generate hypoxic air in a manner similar to the CAT system. Simulated altitudes of about 21,500 feet can be achieved with the Hypoxico Altitude Training Systems.
  • a hypoxia induction system intakes ambient air, adds nitrogen to the intake air, to produce the oxygen-depleted air that is delivered to the subject.
  • the nitrogen can provided from, e.g., liquid nitrogen, cylinders of highly purified nitrogen (e.g., 100% nitrogen), or cylinders containing nitrogen (e.g., 80-95%) and oxygen (e.g., 5-20%).
  • Nitrogen used to produce oxygen-depleted air can optionally be produced by pressure swing adsorption (PSA). Under high pressure, gases tend to be adsorbed to solid surfaces. When the pressure is reduced, the gas is desorbed.
  • PSA pressure swing adsorption
  • Dual step PSA can be used to produce a highly pure nitrogen gas: first, compressed air is forced through a carbon molecular sieve to produce nitrogen at a purity of approximately 98%; second, the nitrogen produced in the first step is forced into a second carbon molecular sieve and the nitrogen gas reaches a purity up to 99.999%. This nitrogen gas can be combined with ambient air to produce oxygen-depleted air that is delivered to the subject.
  • the therapeutic gas delivered to the subject can contain between 5 to 20% O 2 .
  • the proportion of oxygen in the therapeutic gas can vary based upon factors including the age of the subject, the condition treated, and the duration of the hypoxic treatment.
  • the therapeutic gas can contain between 5 to 20% O 2 , between 6 to 18% O 2 , between 7 to 17% O 2 , between 8 to 16% O 2 , between 9 to 15% O 2 , between 10 to 15% O 2 , between 10 to 14% O 2 , between 10 to 13% O 2 , between 10 to 12% O 2 , or between 10 to 11% O 2 .
  • the therapeutic gas contains about 5% O 2 , about 6% O 2 , about 7% O 2 , about 8% O 2 , about 9% O 2 , about 10% O 2 , about 11% O 2 , about 12% O 2 , about 13% O 2 , about 14% O 2 , about 15% O 2 , about 16% O 2 , about 17% O 2 , or about 18% O 2 .
  • the therapeutic gas can be administered to the subject by devices including but not limited to nasal prongs, a face mask, an enclosed tent or chamber (completely or semi-sealed), an intra-tracheal catheter, an endotracheal tube, or a tracheostomy tube.
  • devices including but not limited to nasal prongs, a face mask, an enclosed tent or chamber (completely or semi-sealed), an intra-tracheal catheter, an endotracheal tube, or a tracheostomy tube.
  • nasal prongs or a face mask allow for greater mobility of the subject while being treated with the therapeutic gas.
  • the therapeutic gas can optionally be inhaled by the subject continuously over the course of a variety of time periods (e.g., at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 15 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 48 hours, or longer).
  • time periods e.g., at least 1 minute, at least 2 minutes, at least 3 minutes, at least 4 minutes, at least 5 minutes, at least 6 minutes, at least 7 minutes, at least 8 minutes, at least 9 minutes, at least 10 minutes, at least 15 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 48 hours, or longer).
  • administration of the therapeutic gas is intermittent (e.g., only during inspiration to save gas (sensing inspiration by a variety of methods, temperature, pressure, etc.)) or only for a few hours each day, wherein the therapeutic gas is inhaled (e.g., for a period of time specified above), followed by breathing ambient air, which is then followed by one or more additional periods of breathing the therapeutic gas.
  • the therapeutic gas can optionally be administered to a subject during sleep. This can be achieved by, for example, positioning a tent over a bed or placing a bed within an enclosed chamber. For children or infants, a tent can be positioned over a crib or a crib can be placed within an enclosed chamber.
  • a subject treated with the therapeutic gas can be monitored to measure the effects mediated by the treatment.
  • arterial oxygen saturation (SpO 2 ) can be measured in the subject one or more times (continuously or intermittently) after administration of the therapeutic gas to the subject.
  • the measured SpO 2 value can be used to feedback and adjust (e.g. automatically adjust) the oxygen content of the administered therapeutic gas so as to maintain SpO 2 in the subject in the range of 50-90% (e.g., 60-90%, 70-90%, 75-90%, 80-90%, 85-90%, or about 85%).
  • arterial partial oxygen pressure (PaO 2 ) can be measured in the subject one or more times (continuously or intermittently) after administration of the therapeutic gas to the subject.
  • the measured PaO 2 value can be used to feedback and adjust (e.g. automatically adjust) the oxygen content of the administered therapeutic gas so as to maintain PaO 2 in the subject in the range of 25 mm Hg to 90 mm Hg (e.g., 25 mm Hg to 70 mm Hg, 25 mm Hg to 60 mm Hg, 25 mm Hg to 55 mm Hg, 25 mm Hg to 45 mm Hg, 25 mm Hg to 40 mm Hg, 25 mm Hg to 35 mm Hg, 35 mm Hg to 70 mm Hg, 35 mm Hg to 60 mm Hg, 45 mm Hg to 60 mm Hg, 50 mm Hg to 60 mm Hg, 55 mm Hg to 60 mm Hg, or about 55 mm Hg).
  • 25 mm Hg to 90 mm Hg e.g., 25 mm Hg to 70 mm Hg, 25 mm Hg to 60 mm Hg, 25 mm Hg to 55 mm Hg
  • SpO 2 and PaOz are both measured in the subject one or more times (continuously or intermittently) after administration of the therapeutic gas to the subject.
  • the measured SpO 2 and PaO 2 values can be used to feedback and adjust (e.g. automatically adjust) the oxygen content of the administered therapeutic gas so as to maintain SpO 2 and PaO 2 in the subject in the range of 50-90% SpO 2 and 25 mm Hg to 70 mm Hg PaO 2 (e.g., 80-90% SpO 2 and 50 mm Hg to 60 mm Hg PaO 2 , 85-90% SpO 2 and 55 mm Hg to 60 mm Hg PaO 2 , or about 85% SpO 2 and about 55 mm Hg PaO 2 ).
  • Blood hematocrit and/or circulating hemoglobin levels can be measured in a subject one or more times after administration of the therapeutic gas to the subject, wherein the detection of an increase in blood hematocrit and/or an increase in circulating hemoglobin levels after initiation of the treatment indicates that the treatment is having its desired effect.
  • a decision of whether or how to continue the treatment can be based upon the measured blood hematocrit and/or circulating hemoglobin levels. For example, if the measured blood hematocrit and/or circulating hemoglobin levels exceed a pre-established target level, then the treatment may be terminated or the oxygen content of the administered therapeutic gas may be increased. In another example, if the measured blood hematocrit and/or circulating hemoglobin levels are below a pre-established target level, then the duration of the treatment may be extended and/or the oxygen content of the therapeutic gas may be decreased.
  • a system for treatment or prevention according to the methods described herein can optionally contain: (i) an enclosed tent or chamber or a breathing apparatus; (ii) a hypoxia induction system that delivers oxygen-depleted air to the enclosed tent or chamber or the breathing apparatus, wherein the oxygen-depleted air comprises between 5 to 20% O 2 (or any of the oxygen concentrations described herein); and (iii) a device (such as a pulse oximeter) that measures SpO 2 in a subject breathing air within the enclosed tent or chamber or from the breathing apparatus, wherein the system adjusts the oxygen content of the oxygen-depleted air delivered to the enclosed tent or chamber or the breathing apparatus based upon the SpO 2 measured by the device such that SpO 2 in the subject is maintained within the range of 50% to 90% (or any of the SpO 2 target ranges or values described herein).
  • a hypoxia induction system that delivers oxygen-depleted air to the enclosed tent or chamber or the breathing apparatus, wherein the oxygen-depleted air comprises between 5 to 20% O 2 (or any of the
  • a system for treatment or prevention according to the methods described herein can optionally contain: (i) an enclosed tent or chamber or a breathing apparatus; (ii) a hypoxia induction system that delivers oxygen-depleted air to the enclosed tent or chamber or the breathing apparatus, wherein the oxygen-depleted air comprises between 5 to 20% O 2 (or any of the oxygen concentrations described herein); and (iii) a device that measures PaO 2 in a subject breathing air within the enclosed tent or chamber or from the breathing apparatus, wherein the system adjusts the oxygen content of the oxygen-depleted air delivered to the enclosed tent or chamber or the breathing apparatus based upon the PaO 2 measured by the device such that PaO 2 in the subject is maintained within the range of 25 mm Hg to 90 mm Hg (or any of the PaO 2 target ranges or values described herein).
  • Mitochondrial dysfunction, an oxidative stress disorder, or an inflammatory disorder can also be treated or prevented by causing a subject to breathe a therapeutically effective amount of air in a hypobaric chamber.
  • the hypobaric chamber has an atmospheric pressure equivalent to that between 1,500 meters to 10,000 meters above sea level (e.g., equivalent to 1,500 meters to 10,000 meters above sea level, equivalent to 2,000 meters to 8,000 meters above sea level, equivalent to 2,000 meters to 7,000 meters above sea level, equivalent to 2,000 meters to 6,000 meters above sea level, equivalent to 2,000 meters to 5,000 meters above sea level, equivalent to 2,000 meters to 4,000 meters above sea level, equivalent to 2,000 meters to 3,000 meters above sea level, equivalent to 2,000 meters to 2,500 meters above sea level, equivalent to 3,000 meters to 7,000 meters above sea level, equivalent to 3,000 meters to 6,000 meters above sea level, equivalent to 3,000 meters to 5,000 meters above sea level, equivalent to 3,000 meters to 5,000 meters above sea level, equivalent to 3,000 meters to 5,000 meters above sea level, equivalent to 3,000 meters to
  • hypoxic breathing according to a method described herein may have adverse effects, such as causing pulmonary vasoconstriction, pulmonary hypertension, or shunting via the ductus arteriosus or a patent foramen ovale.
  • gaseous nitric oxide can optionally be administered to a subject in combination with breathing a therapeutic gas or breathing air in a hypobaric chamber to produce systemic hypoxia according to a method of treatment or prevention described herein. See Frostell et al. (1993) Anesthesiology 78:427-35 .
  • nitric oxide can optionally be administered within the therapeutic gas having reduced oxygen content.
  • lower body SpO 2 e.g., toe
  • nitric oxide is added to the inhaled therapeutic gas in an amount that causes the lower body SpO 2 to increase so as to decrease or eliminate the difference between lower body SpO 2 and upper body SpO 2 (e.g., as measured in a finger or ear).
  • nitric oxide for inhalation is available commercially (INOmax TM , Mallinckrodt Pharmaceuticals).
  • the concentration of gaseous nitric oxide in the therapeutic gas administered to the subject can be, for example, at least 5 ppm, at least 10 ppm, at least 20 ppm, at least 40 ppm, at least 50 ppm, at least 80 ppm, at least 100 ppm, at least 200 ppm, at least 300 ppm, or at least 500 ppm.
  • the concentration of gaseous nitric oxide in the therapeutic gas is in the range of 0.5 ppm to 500 ppm (e.g., 0.5 ppm to 200 ppm, 0.5 ppm to 80 ppm, 1 ppm to 200 ppm, 1 ppm to 100 ppm, 1 ppm to 50 ppm, or 5 ppm to 40 ppm).
  • Gaseous nitric oxide can be administered by inhalation from a source of stored, compressed nitric oxide gas.
  • the source of nitric oxide can be 100% nitric oxide, or diluted with N 2 or any other inert gas (e.g., helium).
  • the nitric oxide can be obtained and stored as a mixture free of any contaminating O 2 or higher oxides of nitrogen, because such higher oxides of nitrogen (which can form by reaction of O 2 with nitric oxide) are potentially harmful to lung tissues. If desired, purity of the nitric oxide may be demonstrated with chemiluminescence analysis, prior to administration to a patient.
  • Chemiluminescence NO-NO x analyzers are commercially available (e.g., Model 14A, Thermo Environmental Instruments, Franklin, MA).
  • the NO-N 2 mixture may be blended with O 2 or an O 2 -containing gas such as air through, for example, calibrated rotameters which have been validated previously with a spirometer.
  • the final concentration of nitric oxide in the breathing mixture may be verified with a chemical or chemiluminescence technique (see, e.g., Fontijin et al., Anal. Chem. 42:575 (1970 )).
  • Nitric oxide gas may also be mixed with room air, using a standard low-flow blender (e.g., Bird Blender, Palm Springs, CA).
  • Nitric oxide may be generated from a mixture of N 2 and O 2 (i.e., air) by using an electric plasma nitric oxide generator.
  • an electric plasma nitric oxide generator Such a generator is described in Zapol, U.S. Patent No. 5,396,882 and US published application number 20160030699 .
  • Xenon upregulates hypoxia inducible factor 1 alpha and can be used to treat mitochondrial dysfunction, an oxidative stress disorder, or an inflammatory disorder according to the methods described herein.
  • xenon can be administered to a subject in combination with breathing a reduced oxygen content therapeutic gas or breathing air in a hypobaric chamber according to a method of treatment or prevention described herein.
  • xenon can optionally be administered within the therapeutic gas.
  • the concentration of xenon (an anesthetic gas with therapeutic properties in ischemia-reperfusion) in the therapeutic gas administered to the subject can be, for example, 20-70% (e.g., 30-60%, 30-50%, 40-50%, 40-50%, about 40%, or about 45%).
  • the term "pharmaceutical composition” refers to a preparation of one or more of the components described herein, or physiologically acceptable salts or prodrugs thereof, with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • prodrug refers a precursor compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide the active compound.
  • excipient refers to an inert or inactive substance added to a pharmaceutical composition to further facilitate administration of a compound.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • compositions of the present invention PHD inhibitors selected from the group consisting of 2-oxoglutarate analogs (also known as ⁇ -ketoglutarate), ⁇ -oxocarboxylic acids, and BAY-85-3934.
  • the PHD inhibitor is FG-4592 or roxadustat.
  • Pharmaceutical compositions can be formulated into a single dosage form. In certain embodiments, this dosage form is an oral dosage form. This oral dosage form can be in the form of tablets, pills, dragees, capsules, liquids (aqueous or non-aqueous solutions), gels, syrups, slurries, gelcaps, lozenges, suspensions, and the like, for oral ingestion by a patient.
  • one or more of the pharmaceutical compositions are in a slow release composition or have been formulated to affect release from the oral dosage form.
  • these dosage forms can be administered by any method known in the art including intravenously and intraperitoneally.
  • compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or by lyophilizing processes.
  • compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more pharmaceutically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the term "administration" or any lingual variation thereof as used herein is meant any way of administration.
  • the pharmaceutical composition may be administered in one therapeutic dosage form or in two separate therapeutic dosages such as in separate capsules, tablets or injections. In the case of the two separate therapeutic dosages, the administration may be such that the periods between the administrations vary or are determined by the practitioner. It is however preferred that the second drug is administered within the therapeutic response time of the first drug.
  • the multiple therapeutic dosage forms may be administered either at the same time, or separately, or sequentially, according to the invention, do not represent a mere aggregate of known agents, but a new combination with the valuable property that the effectiveness of the treatment is achieved at a much lower dosage of said at least one additional drug.
  • compositions of the present invention may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with any other therapeutic agent. Administration can be systemic or local.
  • compositions of the invention include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically to the cars, nose, eyes, or skin.
  • the preferred mode of administration is left to the discretion of the practitioner, and will depend in part upon the site of the medical condition (such as the site of cancer) and the severity of thereof.
  • composition of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants for example DMSO, or polyethylene glycol are generally known in the art.
  • the composition can be formulated readily by combining the active components with any pharmaceutically acceptable carriers known in the art.
  • carriers may facilitate the manufacture of such as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose, and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • PVP polyvinylpyrrolidone
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active components may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different dosages or dosage forms.
  • stabilizers may be added.
  • compositions for parenteral administration include aqueous solutions of the active preparation in a water-soluble form. Additionally, suspensions of the active preparation may be prepared as oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl, cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds, to allow for the preparation of highly concentrated solutions.
  • the composition may be in a powder form for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.
  • a suitable vehicle e.g., sterile, pyrogen-free water.
  • the exact formulation, route of administration and dosage may be chosen by the physician familiar with the patient's condition. (See for example Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Chapter I, p. 1 ).
  • dosing can also be a single administration of a slow release composition, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • Mitochondrial disease was modeled in the suspension cell line, K562, and a CRISPR screen was performed to identify potential drug targets.
  • the natural product, Antimycin was used as a complex III inhibitor of the respiratory chain. In the presence of Antimycin, the respiratory chain is unable to oxidize high energy reducing equivalents to power ATP production. However, redox recycling by the cytoplasmic lactate dehydrogenase reaction, allows other NAD+ dependent reactions to proceed. Removal of pyruvate in the presence of respiratory chain inhibition resulted in reductive stress, further preventing cell proliferation ( King, Science, 246:500-03 (1989 )).
  • Mitochondrial disease was modeled with the addition of Antimycin alone (moderate disease) or Antimycin in combination with removal of pyruvate (severe disease), using cell growth as a proxy for disease magnitude ( FIG. 3 ).
  • K562 cells were infected with a ⁇ 65,000 sgRNA library, targeting ⁇ 18,000 genes ( Shalem et al., Science, 343:84-88 (2014 )).
  • the pool of knockout cells were divided into experimental conditions of untreated, moderate disease and severe disease states ( FIG. 4 ). Samples for an enrichment screen were collected by allowing the knockout pool to grow in selection conditions for three weeks. The relative growth between untreated and moderate disease conditions was 100-fold and between untreated and severe disease conditions was 10,000-fold ( FIG. 5 ).
  • VHL Von Hippel Lindau
  • RIGER RNAi Gene Enrichment Ranking
  • VHL knockout cells were also enriched in untreated conditions over time, reflecting an overall effect on cell growth. However, this enrichment was significantly less than in disease conditions. Table 2. Enrichment of sgRNAs and corresponding genes based on CRISPR in disease conditions relative to pre-treatment conditions.
  • VHL activity is a key regulator of the hypoxia response pathway ( Ohh et al., Nature Cell Biol. 2:423-27 (2000 )). Organisms have evolved elaborate defense mechanisms to cope with changing oxygen tensions and extreme environments. In normoxic conditions, the hypoxia inducible transcription factors (HIF) are constitutively made and hydroxylated by the prolyl-hydroxylase (PHD) enzymes ( FIG. 1 ) ( Majmunder et al., Molecular Cell. 40:294-309 (2010 )). The hydroxylated form is recognized by the ubiquitin ligase, VHL, and targeted for degradation. During hypoxia, the PHD reaction does not take place, allowing HIP1 ⁇ stabilization and activation of the hypoxia transcriptional program.
  • HIP1 ⁇ prolyl-hydroxylase
  • VHL-knockout cells show HIF1 ⁇ stabilization, even during normoxic conditions, thereby bypassing cellular oxygen sensing mechanisms ( Ivan et al., Proc. Natl. Acad. Sci. U.S.A. 99:13459-64 (2002 )).
  • VHL was validated and characterized as a therapeutic target by testing the ability of VHL-knockout cells to withstand respiratory chain dysfunction.
  • VHL-modified cells showed increased cell proliferation in the presence of Antimycin (moderate and severe disease conditions) relative to non-targeting (dummy) sgRNA-modified cells ( FIG. 11 ).
  • Antimycin moderate and severe disease conditions
  • non-targeting dummy sgRNA-modified cells
  • the therapeutic value in the context of complex III deficiency could be extended to lesions in other mitochondrial complexes.
  • VHL-knockout cells were also more resistant to Complex I inhibition by Piericidin and ATP synthase inhibition by Oligomycin, demonstrating the broad utility of this therapeutic approach.
  • mitochondrial disease encompasses such a diverse set of genetic lesions impacting the respiratory chain complexes in varying combinations, such a generic therapeutic is especially attractive.
  • a genome-wide CRISPR screen identified the hypoxia response pathway as the strongest suppressor of mitochondrial disease.
  • the genome-scale CRISPR knockout (GECKO) library v1 was generously provided by the Zhang Lab. Library details have previously been published ( Shalem et al., Science, 343:84-88 (2014 )).
  • 1.2e7 cells were placed in each of 25, T225 flasks in 50ml of full DMEM (Life Technologies 11995) media (containing Pen/Strep, 10% FBS). 18h later, media in each flask was replaced with 13ml of DMEM (no Pen/Strep, 10% FBS) and 2h later, media was replaced with 20ml OptiMEM (Life Technologies 31985-070; no serum, no Pen/Strep).
  • a transfection mastermix was made by combining individually prepared mastermix A (94ml of OptiMEM, 2.4ml of Lipofectamine 2000 (Life Technologies)) and mastermix B (94ml OptiMEM, 2.1ml of PLUS Reagent (Life Technologies), 240 ⁇ g of pVSVg plasmid, 360 ⁇ g of psPAX2 plasmid and 480 ⁇ g of GECKO plasmid library). Mastermixes A and B were combined at RT for 20m. 8ml of the final mastermix was added to each T225 flask of HEK 293 cells.
  • DMEM media w/ 1% BSA (Sigma)
  • Virus was concentrated by spinning for 2h at 24,000 rpm using a SW32Ti rotor. Virus was resuspended overnight at 4C, allowing pellets to dissolve. Library virus was stored at -80C prior to use.
  • K562 cells were obtained from ATCC and maintained in full DMEM media (10% FBS, Pen/Strep). K562 cells were grown in 1L spinner flasks (Matrical) on magnetic stir plates (Bellco). Cells were always passaged before reaching confluency (1e6/ml) and subcultured at a concentration of 1e5/ml.
  • 250e6 K562 cells were resuspended to a concentration of 1.5e6 cells/ml.
  • Polybrene Sigma
  • 2ml of this cell suspension was placed in each well of 5, 12-well plates.
  • 10ul of virus was added to each well for a target MOI of 0.3, ensuring that most cells incorporated 1 or 0 lentivirus particles. Plates were spinfected for 2h at 1000g and placed in an incubator for 1h, after which media was aspirated.
  • 2ml of full DMEM media (10% FBS, Pen/Strep) was placed in each well and cells were resuspended.
  • Infected cells were passaged before reaching 1e6/ml and maintained in Puromycin-containing conditions for one week after infection. At this point, 70e6 cells were pelleted and stored as pre-treatment (Early) samples for each infection replicate.
  • Sequencing libraries were prepared as previously described ( Shalem et al., Science, 343:84-88 (2014 )). Briefly, DNA was extracted using the Qiagen Blood and Cell Culture DNA Maxi Kit from 70e6 cells per experimental condition, for each infection replicate. DNA was then purified using Micro Bio-Spin columns (BioRad 732-6224). 25 PCR reactions were performed using Herculase II Fusion DNA Polymerase (Agilent) to amplify the sgRNAs from genomic DNA, at a minimum coverage of 450x per sgRNA. 30ul from the first pooled PCR samples were used as input for the second PCR reaction, allowing for attachment of barcodes and sequencing adapters.
  • Custom Python and Matlab scripts were written for processing of sequencing reads. Reads were trimmed to remove barcodes and sequences corresponding to the GECKO library backbone. A custom bowtie library was created for mapping between sgRNA sequences and guide/gene names. Bowtie alignment was performed, allowing for single mismatches. Finally, guide abundance was compiled for each experimental condition and a matrix mapping guide name to abundance for all samples was created.
  • fold-enrichment was calculated for moderate or severe disease conditions relative to pre-treatment conditions. The most enriched genes were then individually checked for their overall effect on cell viability (untreated relative to pre-treatment conditions). Alternatively, fold enrichment was also calculated for untreated vs. disease conditions. However, this form of analysis confounds genes which are enriched in disease conditions or selectively depleted in untreated conditions. The top hit was found using either approach.
  • RIGER analysis (Luo et al., 2008) was used to generate a summary statistic by combining information corresponding to all sgRNAs for a given gene. SgRNAs were pre-ranked by fold-enrichment between two conditions. The Kolmogorov-Smirnov method was used with 1000 permutations. Gene scores were not adjusted for the number of sgRNAs corresponding to a given gene.
  • VHL was confirmed as an effective therapeutic target for mitochondrial disease
  • clinically-relevant options for triggering the hypoxia response were studied. While a VHL-inhibitor has been reported ( Buckley et al., J. Am. Chem. Soc. 134:4465-68 (2012 )), it is not cell permeable.
  • PHD inhibitors have been extensively developed for the treatment of anemia of chronic kidney disease and ischemic injury ( Rabinowitz, J. Med. Chem. 56:9369-9402
  • FG-4592 The small molecule PHD inhibitor, FG-4592, also known as roxadustat, is currently in Phase III clinical trials for chronic kidney disease.
  • FG-4592 shows efficacy in treatment of kidney disease by upregulating the canonical marker of the hypoxia response, erythropoietin (EPO).
  • EPO erythropoietin
  • FG-4592 treatment may mimic VHL-knockout, thus triggering a broader hypoxia transcriptional program.
  • Compounds that trigger the hypoxia response including VHL-inhibitors and PHD inhibitors are therapeutics for mitochondrial dysfunction. Pre-treatment and incubation with FG-4592 almost completely rescued the growth defects caused by deficiency of multiple respiratory complexes, in multiple cell lines ( FIGs. 12-13 ).
  • the rescue mechanism of FG-4592 was characterized by studying its effect on the hypoxia response and energy metabolism. While HIF1 ⁇ is undetectable during normoxic conditions, treatment with FG-4592 stabilized the transcription factor even during normoxia. In hypoxic conditions, FG-4592 stabilized HIF1 ⁇ beyond the endogenous hypoxia response of untreated cells. It has previously been noted that a paradox exists between mitochondrial dysfunction and cellular sensing of hypoxia ( Chandel et al., Proc. Natl. Acad. Sci. U.S.A. 95:11715-20 (1998 )). In cell culture, inhibition of the respiratory chain prevents HIF stabilization, even under low oxygen conditions that would otherwise trigger the hypoxia response.
  • the HIF transcriptional response is believed to be protective during states of hypoxia, at least in part by causing a rewiring of energy metabolism.
  • the HIF1 ⁇ response can maintain the energy supply at low oxygen tensions.
  • FG-4592 caused normoxic stabilization of HIF proteins.
  • Glycolytic enzymes such as hexokinase 2 (HK2) and glucose transporter 1 (GLUT1) were upregulated in multiple cell types. Glucose import and phosphorylation are rate-limiting steps in the glycolytic flux of many tissues.
  • Lactate dehydrogenase maintains the redox balance during respiratory chain inhibition and was similarly upregulated with FG-4592 treatment.
  • HIF1 ⁇ activation is also known to shunt the carbon supply away from the TCA cycle and towards the LDH reaction.
  • Several mechanisms have been proposed for this shift away from aerobic respiration, including pyruvate dehydrogenase phosphorylation.
  • PDK1 pyruvate dehydrogenase kinase
  • none of the aforementioned enzymes were upregulated by respiratory chain inhibition alone. Thus, cells were unable to mount the appropriate transcriptional response by endogenous mechanisms.
  • FG-4592 treatment triggered the hypoxia transcriptional program, even during respiratory chain inhibition ( FIG. 15 ).
  • HIF pathway Genetic and small molecule activation of the HIF pathway enabled cells to cope with mitochondrial dysfunction at multiple steps of the electron transport chain and in multiple cell types.
  • a zebrafish reporter strain was used to monitor activation of the hypoxia transcriptional response ( Santhakumar et al., Cancer Res. 72, 4017-27 (2012 )).
  • the reporter fish has a promoter containing hypoxia response elements (HREs) fused to GFP.
  • Hypoxia exposure or VHL knockdown causes an increase in GFP expression, allowing for in vivo monitoring of the hypoxia response.
  • This model system could be used to monitor drug-target engagement and pharmacodynamics of a HIF activator ( Chowdhury et al., ACS Chem Biol. 8:1488-96 (2013 )).
  • zebrafish embryos have been shown to have a dose-responsive sensitivity to multiple, specific respiratory chain inhibitors including Antimycin ( Pinho et al., Br. J. Pharmacol. 169:1072-90 (2013 )).
  • FG-4592 treatment resulted in a time-dependent increase in fluorescence of individual reporter fish. After even 6 hours of exposure to 2.5 ⁇ M FG-4592, a robust increase in fluorescence was detected ( FIG. 16 ). Thus, FG-4592 engaged the zebrafish prolyl-hydroxylases and triggered the hypoxia transcriptional program. Antimycin inhibits oxygen consumption of zebrafish embryos ( Stackley, et al., PLOS One. 6, e25652 (2011 )). Prolonged treatment of Antimycin at low nanomolar concentrations resulted in death of embryos. Antimycin-induced death of zebrafish embryos was prevented by treating the fish with FG-4592. At intermediate concentrations of Antimycin, FG-4592 treatment nearly doubled survival ( FIG. 17 ). As both the hypoxia response pathway and mitochondrial functions are particularly well conserved, this is expected to be relevant for higher organisms.
  • Example 5 Hypoxia as a Therapeutic in a Mouse Model of Leigh Syndrome
  • hypoxia response as a suppressor of mitochondrial dysfunction.
  • chronic exposure to moderate environmental hypoxia - at 11% O 2 a level known to be tolerated in humans - was evaluated to determine whether it could alleviate the disease phenotype in a genetic mouse model of mitochondrial disease.
  • Leigh syndrome is the most common pediatric form of mitochondrial disease. Though relatively healthy at birth, patients develop irreversible neurodegeneration by two years of age ( Lake et al., Journal of Neuropathology & Experimental Neurology. 74(6), 482-492 (2015 )). These patients suffer deep, bilaterally symmetric lesions in the gray matter of the brain stem and basal ganglia, with marked gliosis. Most patients die between the ages of 3-16 months. To date, over 60 different genes have been identified that can underlie this devastating syndrome, with Complex I deficiency being the most frequent biochemical cause of disease.
  • a mouse model of Leigh Syndrome has been generated by disruption of the murine Ndufs4 gene ( Kruse et al., Cell metabolism. 7(4), 312-320 (2008 )). Loss of NDUFS4 in humans leads to one of the more severe recessive forms of Leigh syndrome.
  • the murine model has been studied at atmospheric (21% O 2 ) levels and faithfully recapitulates much of the reported human disease phenotype.
  • Ndufs4 knockout (KO) mice display retarded growth rates, have impaired visual acuity, and have a delayed startle response. Their body temperature falls progressively until reaching 32°C, shortly before death at 50-60 days of age. Diseased mice also display locomotor deficits and failure to thrive by 50 days. Their neuro-histopathology closely resembles clinical findings, with a substantial inflammatory response in the brainstem and cerebellum. Since this mouse model is well characterized and representative of mitochondrial disease progression, chronic hypoxic exposure was tested as a therapeutic strategy in this model.
  • Ndufs4 KO mice were first studied to determine whether they were able to tolerate brief hypoxic exposure and mount a hypoxic response in a manner similar to wild type (WT) mice.
  • WT mice and three KO mice were exposed to breathing 8.5% oxygen at sea level pressure for 6 hours. This gave the mice sufficient time to mount a transcriptional and translational response.
  • Acute exposure of WT mice to hypoxia triggers HIF stabilization, resulting in Epo transcription and translation.
  • Epo protein levels in plasma were measured and showed that both WT and KO mice upregulated Epo production to a similar extent, increasing circulating EPO levels by approximately 40-fold.
  • Mice were placed in 60 liter plexiglass chambers that were given a gas mixture of compressed air and 100% N 2 , compressed air alone, or compressed air and 100% O 2 (Airgas Inc.). The gas flow rates were measured and controlled with rotameters and valves. Oxygen concentrations were measured several times each day at the outlet of the chambers using an oxygen analyzer (MiniOx I Oxygen Analyzer, Ohio Medical Corporation) and the flow rates of air, nitrogen and oxygen were modified if necessary in order to obtain a stable oxygen concentration of 11% in the hypoxic chamber (and 55% in the mild-hyperoxia chamber).
  • an oxygen analyzer MiniOx I Oxygen Analyzer, Ohio Medical Corporation
  • Soda lime (Sodasorb, Smiths Medical) (approximately 250g), was placed on the floor of each chamber to scavenge carbon dioxide (CO 2 ) produced by the animals and replaced every 3 days.
  • CO 2 carbon dioxide
  • the total flow of fresh gas flushing each chamber was adjusted between 5 and 10 L/min to maintain the chamber CO 2 level below 0.4% and the relative humidity between 30% and 70%. Mice were exposed to gas treatment continuously for 24 hours per day, 7 days a week.
  • Ndufs4 KO and control mice were continuously exposed to normoxia or hypoxia (11% O 2 ) after enrollment in the experiment, excluding temporary removal for behavior tests and maintenance three times per week.
  • Untreated Ndufs4 KO mice typically begin to show significant disease progression after approximately 30 days of post-birth air exposure, which is about ten days after weaning. Since hypoxia-related vascular responses (constriction of pulmonary circulation, dilation of ductus arteriosus) occur in early post-natal development, chronic hypoxic exposure treatments were initiated when mice were 30 days old.
  • mice showed a striking rescue in all aspects of health that were tested, including body weight gain, core temperature maintenance, and neurologic behavior.
  • All Ndufs4 KO mice continued to gain weight between 30-37 days of age ( FIGS. 18B-C ). At this stage, untreated KO mice lost weight, became hypothermic, and died. In contrast, Ndufs4 KO mice breathing 11% O 2 gained weight for several weeks, at which point body weight gain slowed, similar to the growth kinetics of WT mice.
  • the growth rate of hypoxia-treated Ndufs4 KO mice matched that of hypoxia-treated WT mice, suggesting that the primary cause of weight loss in Ndufs4 KO mice was alleviated by hypoxic exposure.
  • Ndufs4 KO mice At 30 days of age, untreated Ndufs4 KO mice have similar core body temperatures to WT mice. By 50 days, there is nearly a 4°C drop in temperature in the Ndufs4 KO mice ( FIG. 18D ). However, Ndufs4 KO mice treated with chronic 11% O 2 breathing showed no reduction of core body temperature ( FIG. 18D ). Thus, chronic hypoxic breathing rescues the underlying metabolic phenotype that directly or indirectly results in alterations of energy and nutrient metabolism.
  • Ndufs4 KO mice exhibit striking defects in locomotor activity. Ataxia and failure to thrive are hallmarks of mitochondria dysfunction. Behavioral tests were performed in 10 day intervals in normoxia and hypoxia-treated, WT and KO mice.
  • the rotarod test ( Caston et al., Neurobiology of learning and memory, 64:195-202 (1995 )) measures the ability of mice to maintain grip strength, balance, and fatigue resistance on an accelerating, rotating rod.
  • a rotarod machine Ugo Basile was used to measure the ability of mice to stay on an accelerating, rotating rod. Rotarod parameters were as follows: acceleration of 5 rpm/m and a maximum speed of 40 rpm.
  • mice On each measurement day, three trials were performed, with individual trials at least 10 minutes apart to allow mice to recuperate. The median time on rotarod is reported. If mice used their body to grasp the rod (rather than walking on it) for more than 10 seconds, this time was recorded as time of fall. Age of measurements is +/- 5 days for practical purposes without any age bias between groups.
  • Hypoxia-treated WT mice performed similarly to normoxia-treated control mice. Remarkably, KO mice breathing 11% O 2 displayed a near complete rescue of this locomotor phenotype.
  • spontaneous locomotor activity was measured as total distance traveled within an hour.
  • the open field instrument Med Associates Inc.
  • Mice were blindly chosen for a given day and placed in open field chambers for 1 hour.
  • Spontaneous locomotor activity was measured based on beam breaks and recorded by the instrument.
  • the traces shown in FIG. 18F are representative of a sick, Ndufs4 KO mouse exposed to 21% O 2 (age of such sickness varies slightly between mice), age-matched KO and WT mice exposed to 11% O 2 , and WT mice exposed to 21% O 2 .
  • Age of measurements is +/- 5 days for practical purposes without any age bias between groups.
  • Untreated KO mice show drastically reduced spontaneous locomotor activity ( FIG. 18F ). This defect was significantly rescued in hypoxia-treated mice, however only to 50% of the values of control mice ( FIG. 18F and 20 ).
  • Example 6 Modest Hyperoxic Exposure is Lethal for a Murine Model of Leigh Syndrome
  • Ndufs4 KO mice were further characterized following treatment with chronic hypoxia.
  • Complex 1 Activity was measured in cerebellum tissue from mice. Tissue was homogenized in 1 ml of ice-cold PBS using a Qiagen TissueLyser II. Approximately 50-100 mg of tissue was used as input material for the Complex I Enzyme Activity Microplate Assay Kit (ab109721, Abcam). Absorbance was read at 450nm wavelength and recorded every 30 seconds for 135 total measurements. Background signal was not subtracted in data shown.
  • the circulating blood hematocrit in the KO mice was elevated from 40% during normoxia to approximately 60% during hypoxia, indicating EPO target engagement by hypoxic breathing ( FIG. 19A ). Furthermore, although Ndufs4 KO mice appear quite healthy following hypoxia treatment, brain Complex I activity remained dramatically reduced to the same levels as untreated Ndufs4 KO mice ( FIG. 19B ).
  • Normoxia-treated KO mice exhibit substantial neuronal degeneration. Lesions are accompanied by Iba-1+ microglial proliferation within olfactory lobes, cerebellum and brainstem as documented elsewhere ( Quintana et al., Proc. Natl. Acad. Sci. U.S.A. 107, 10996-11001 (2010 )). Mice were anesthetized and the chest cavity was opened and a catheter was placed in the left ventricle. The whole body was perfused with ice cold PBS and then with 4% PFA. The brain was dissected out, stored overnight in 4% PFA and then placed in 30% sucrose (in PBS) for two days.
  • Formalin-perfused brains were sectioned parasagittally at the olfactory lobes. Two transverse sections of cerebellum and brainstem were also collected: a rostral section with subjacent pons, and a more caudal section with medulla oblongata. Immunohistochemistry was performed on adjacent tissue sections using an antibody recognizing the microglial marker Iba-1 (Wako; 2 ⁇ g/ml). In contrast to normoxia-treated KO mice, KO mice breathing 11% O 2 exhibited minimal to no lesions (FIG. 19C ), and were virtually indistinguishable histologically from WT controls.
  • ⁇ -hydroxybutyrate has been identified as a circulating plasma marker of Leigh syndrome ( Legault et al., Cell reports. 13, 981:89 (2015 )). Lactate and ⁇ -hydroxybutyrate were quantified in mouse plasma by spiking in each corresponding isotope labeled standard (CDN isotope). A series of standard solutions of metabolites at seven different concentrations were prepared in a surrogate matrix buffer (PBS buffer with 30g/L human serum albumin). 30uL of the mouse plasma sample were combined with 20uL of isotope labeled internal standard, vortexed for 10 seconds and spun down for 10 seconds. Metabolite extractions were performed using 70% acetonitrile.
  • the Xbridge amide HILIC column (2.1 X100 mm, 2.5 ⁇ M particle size, from Waters 186006091) was used to separate metabolites and MS was acquired under the negative ionization mode. The column was maintained at 27°C during runs.
  • the mobile phase A was 20mM ammonium acetate, 0.25 % ammonium hydroxide pH adjusted to 9.
  • the mobile phase B was 100% acetonitrile.
  • the MS data acquisition was full scan mode in a range of 70-1000 m/z, with the resolution set at 140,000, the AGC target at 3E6, and the maximum injection time at 400 msec.
  • ⁇ -hydroxybutyrate was elevated in air-breathing Ndufs4 KO mouse plasma ( FIG. 19D ).
  • Treatment with chronic hypoxia rescued the elevation of this disease marker ( FIG. 19D ).
  • plasma lactate levels were increased in Ndufs4 KO mice breathing air between 50-65 days of age, while this was partially rescued by 11% hypoxic exposure ( FIG. 19E ).
  • these laboratory and histopathological studies confirm that chronic hypoxic exposure to breathing 11% O 2 activates the endogenous hypoxia response. Hypoxia does not fix the proximal lesion within mitochondrial Complex I, but rather, prevents the onset of subsequent biochemical and histopathological defects.
  • Ndufs4 mice were chronically exposed to hypoxia treatment starting at 30 days of age, termed early-stage treatment.
  • Ndufs4 mice were chronically exposed to hypoxia beginning at 55 days of age, termed late-stage treatment.
  • Early-stage treatment began prior to the development of any disease symptoms, allowing for the prevention of disease.
  • Late-stage treatment began after the disease had already manifested, thus testing for the reversal of disease.
  • the early-stage hypoxia treatment group weighed 14.2g, due to the ameliorative effects of hypoxia.
  • normoxic mice of this age weighed 10.6g.
  • Chronically exposing these mice to late-stage hypoxia resulted in an improvement of body weight and body temperature trajectory.
  • the body weights of early-stage treatment and late-stage treatment groups were indistinguishable ( Fig. 21A ).
  • Core body temperature in normoxic mice was 33.5°C at 55 days of age.
  • Late-stage hypoxic treatment resulted in an increase of core temperature from 33.5°C to 35.3°C within five days of hypoxia exposure (p ⁇ 0.05) ( Fig. 21B ).
  • Leigh syndrome patients develop symmetric, bilateral lesions in the brain.
  • the same mode of pathogenesis has previously been demonstrated in normoxic Ndufs4 KO mice.
  • sequential brain MRI scans were performed in four Ndufs4 KO mice receiving late-stage hypoxia therapy. After the first scan, mice were treated with hypoxia for two weeks and a second scan was performed ( Fig. 22 ). After four weeks of 11% O 2 breathing, a third scan was performed.
  • Neuroimaging demonstrated a progressive reduction of intensity and size of lesions in the brainstem and olfactory bulbs, which is observable after the first two weeks of treatment.
  • the IV ventricle appears more diffuse, likely as a consequence of parenchymal atrophy. This abnormal imaging morphology is reversed upon hypoxia treatment. The aforementioned disease pattern was reversed in all four mice that were studied.
  • hypoxia-treated mice remained alive at 170 days of age. Hypoxic exposure was continued to determine the full extent of the survival rescue. 50% survival was observed at 270 days of age. Thus, survival duration of hypoxic rescue treatment was nearly 5-fold that of normoxic KO mice, however it was not a complete rescue relative to wild-type survival. In order to understand the cause of death in this cohort, we began by studying the neurological status of the old, hypoxia-treated mice to determine whether the original disease had manifested.
  • Immunostaining for the inflammatory marker Iba-1 was performed to determine whether hypoxia delays death by delaying neuropathology. Both the cerebellum and olfactory bulb display significant microglial activation in sick, normoxic KO mice. However, none of the hypoxia-treated mice that were greater than 200 days of age displayed any evidence of neuroinflammation by histopathology ( Fig. 23A ). T2-weighted MRI imaging was also performed to detect the neurological lesions. Mice breathing normoxia at 60 days developed lesions in the postero-lateral portion of the brainstem, localized in the vestibular nuclei ( Fig. 23B ).
  • hypoxia-treated mice have neurological lesions that were detectable by MRI.
  • hypoxia appears to reverse neurological lesions as detected by MRI and histopathology, even at greater than 200 days of age. This suggests that death in old, hypoxia-treated KO mice is likely to be attributable to other organ dysfunction (e.g., myocardial failure).
  • Cardiomyopathy is a common presentation of mitochondrial disease.
  • the acute nature of death in the old, hypoxia-treated mice suggested that cardiac dysfunction may play a role in the pathology.
  • a series of echocardiograms was obtained during air breathing to investigate the cause of death in 250 day-old Ndufs4 KO mice breathing hypoxia.
  • LV myocardial contractility was studied and found to be significantly impaired in older, KO mice treated with chronic hypoxia but not in WT controls treated with chronic hypoxia.
  • LV Fractional shortening was 37% versus 60% in WT controls ( Fig. 24B ).
  • echocardiograms were strongly suggestive of chronic pericardial effusions and numerous arrhythmias were noted during the scans.
  • Ndufs4 KO mice were assessed breathing 11% oxygen for ten hours per day from 9am to 7pm, and breathing at normoxia for the remaining 14 hours/day. Given that mice are nocturnal creatures, in this protocol they were breathing normoxia while awake and hypoxia while asleep. This intermittent exposure to hypoxia increased hematocrit to 55% within three weeks, indicative of sufficient physiological adaptation to hypoxia ( Fig. 25E ). However, survival rates were identical to untreated (normoxic) KO mice (Fig. 25A). During exposure to intermittent hypoxia, body weight decreased until either a natural death or humane euthanasia was administered. Mice undergoing the intermittent protocol presented at 60 days with a lower body weight compared to normoxic KO controls ( Fig. 25B ).
  • KO mice were briefly exposed to normoxia 3-5 times per week to allow for maintenance of their cages and behavior studies. These mice never developed brain lesions by neuropathology. Thus, some but not all intermittent hypoxia/normoxia regimens are effective.
  • hypoxic breathing at 11% oxygen is equivalent to breathing the partial pressure of oxygen found at 4,500m of altitude. A very mild level of hypoxia was also assessed, equivalent to 1,500m of altitude. Breathing 17% O 2 did not prevent neurological disease in Ndufs4 KO mice. The clinical manifestations were similar to Ndufs4 KO mice breathing normoxic air. A loss in body core temperature was recorded from 36.5°C to 35.9°C at 50 days of age ( Fig. 26C ). Significant weight loss was also measured ( Fig. 26B ). After 3 weeks of chronic 17% oxygen exposure, the tail venous blood hematocrit was 51% (compared to 60% after three weeks of 11% oxygen exposure) ( Fig. 26D ).
  • Mitochondrial disease patients are particularly susceptible to infections. It is believed that that the hyperimmune response of mitochondrial disease patients may contribute to disease pathogenesis. Furthermore, the mouse model of Leigh Syndrome shows a significant inflammatory response in the brain regions that ultimately suffer from neurodegeneration and cause death by respiratory failure. We investigated whether the Ndufs4 KO mouse model demonstrated increased susceptibility to infection, using LPS as the immune trigger.
  • hypoxia rescues disease symptoms we investigated whether hypoxia protects against the pathology associated with the hyperimmune response in KO mice. Indeed, acute hypoxic exposure of LPS-treated KO mice improved survival duration nearly 3-fold ( Fig. 28A ). Body temperature loss upon LPS treatment was not significantly rescued by hypoxia treatment, as hypoxia itself causes acute changes in body temperature in both WT and KO mice ( Fig. 28B ). This finding indicates that the therapeutic effects of hypoxia may involve an ability to suppress the toxic effects of the hyperimmune response in mitochondrial disease mice. As the inflammatory response involves a significant oxidative stress component, this is a prime example of the therapeutic effect of hypoxia in the setting of an oxidative stress pathology.
  • the present invention further relates to the following items:

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